CN108474005A - Indexable subsystem, kit and application thereof - Google Patents
Indexable subsystem, kit and application thereof Download PDFInfo
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- CN108474005A CN108474005A CN201680071463.4A CN201680071463A CN108474005A CN 108474005 A CN108474005 A CN 108474005A CN 201680071463 A CN201680071463 A CN 201680071463A CN 108474005 A CN108474005 A CN 108474005A
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Abstract
The content of present invention is about a kind of in the method that one foreign DNA sequences are in vitro integrated into cellular genome using an indexable subsystem.The indexing subsystem includes a first vector, carries inverted repeats and an exogenous DNA；And one can express translocase Second support.The content of present invention also provide it is a kind of comprising this to the kit for the indexable subsystem that an exogenous DNA is integrated into cellular genome；And a kind of method to treat individual in need after so that it is carried foreign DNA sequences, then administers an a effective amount of modified immunocyte including an immunocyte is transformed using the method for the present invention and/or kit to the individual.
Description
Technical field
The content of present invention is so that cell is carried system, kit and the method for specific gene about to engineered cells；With
And using these modified cells as healing potion, to treat the purposes of individual in need.
Background technology
PiggyBac (PB) transposon (transposon) is a kind of moveable genetic module (mobile genetic
Element), can efficiently be moved between carrier and genome (genome) by " cliping and pasting " mechanism.In moving process,
PB translocases can recognize the transposon particular inverse terminal repeat (transposon- positioned at two end of indexable subcarrier
Specific inverted terminal repeat sequences, ITRs), so content is effective by home position
(integrate) is integrated to rate to TTAA genomic locations.The activity of PB indexing subsystems can make two positioned at PB carriers
Specific gene between ITRs is moved to easily in target genome.
The characteristics of PB transposons includes：(1) without the length limitation for carrying gene；Known PB transposons can promote to be up to
The DNA fragmentation of 100kbp (kilobase) is moved in target cell；(2) transposition has invertibity；It can will only express PB translocases
Carrier temporarily transfect to comprising be inserted into PB carriers genome in, can be by transposon by being removed in genome；And (3)
PB transposons can be widely applied to different plant species；There is the PB transposons of specificity can carry out gene to various species TTAA to change
It makes, for example, insect cell and mammalian cell.In addition, compared to viral vectors, the immunogenicity of PB transposons
(immunogenicity) relatively low, it is less susceptible to cause allergic reaction.
Immune system is particularly important to human health.Defect occurs for immune system or can not normally act on to reduce individual and disappear
It goes out the ability of abnormal body cell and invasive pathogen, in turn results in the diseases such as tumour or infection.On the other hand, work as immune system
When overactivity, then autologous tissue can be attacked and be injured, cause autoimmune disease and inflammatory response.Therefore, regulation and control are immune thin
Expression, function or the effect (such as being transferred to (introduce) allogenic gene) of born of the same parents, promotes with (i) or reduces immunocyte
Gene expression, or (ii) increase or inhibit the function of immunocyte, provide one kind to prevent and/or treat immune related disease
The method of disease.However, immunocyte can not the characteristic of Stable Expression of Exogenous gene often limit it in terms of the cell therapy
Application.
Therefore, there is an urgent need for a kind of improved expression systems and method for related field, so that immunocyte is effectively and stably
Foreign DNA sequences (such as gene) are expressed, and then applied to cell therapy to prevent or treat immune correlated disease.
Invention content
Invention content be intended to provide the content of present invention simplify abstract so that reader have to the content of present invention it is basic
Understand.The invention content is not the complete overview of the content of present invention, and its be not intended to point out the embodiment of the present invention it is important/
Key component defines the scope of the present invention.
The content of present invention is about a kind of in the side for the genome that a foreign DNA sequences are in vitro integrated into a cell
Method.This method includes：(i) an indexable subsystem is obtained, it includes：(a) first vector, including one by SEQ ID No:1 institute
First inverted repeat of the nucleotide sequence of composition；Foreign DNA sequences positioned at the first inverted repeat downstream；And one be located at
The foreign DNA sequences downstream and include by SEQ ID No:Second inverted repeat of 2 nucleotide sequences formed；And
(b) Second support, including an operatively be connected to the promoter of a nucleic acid, wherein the nucleic acid codified, which goes out one, has
SEQ ID No:The translocase of 4 amino acid sequence；And first and second carrier is transferred to the cell by (ii).Wait for translocase in
After being expressed in the cell, you can the reaction that catalysis is shear off foreign DNA sequences by first vector, and it is thin to be integrated into this
The genome of born of the same parents, foreign DNA sequences are integrated into the genome of cell.
The content of present invention is related to one a foreign DNA sequences to be integrated into the kit of cellular genome.The reagent
Box includes (i) container；(ii) an indexable subsystem, including (a) first vector, including one by SEQ ID No:1 is formed
Nucleotide sequence the first inverted repeat, one is located at the downstream of first inverted repeat and with by SEQ ID No:2 groups
At nucleotide sequence the second inverted repeat and one between first inverted repeat and second inverted repeat, can
Make the cloning site that foreign DNA sequences are transferred to；And (b) Second support, including an operatively be connected to a nucleic acid
Promoter, wherein the nucleic acid codified go out one have SEQ ID No:The translocase of 4 amino acid sequence；And (iii) one
The operating instruction of accompanying, to indicate how using the indexable subsystem of the present invention.The translocase can be catalyzed and be sheared down by first vector
The reaction of foreign DNA sequences, and be integrated into the genome of cell.
In addition, the content of present invention further relate to it is a kind of to treat one suffer from or it is doubtful suffer from immune correlated disease individual
Method.This method includes using ex vivo methods of the present invention (preferably utilizing kit of the present invention), by a foreign DNA sequences
It is integrated into the genome of an immunocyte, the immunocyte is transformed；It is a effective amount of aforementioned engineered to individual administering one again
Immunocyte, to improve or slow down the symptom of immune correlated disease.
After refering to following description, those skilled in the art are when the essence spirit and other that can will readily appreciate that the present invention
Goal of the invention and the technology used in the present invention means and embodiment.
Description of the drawings
Figure 1A is a block diagram, be about will be sheared with enzyme/helper plasmid for preparing of connection method and DNA donors it is (cyclic annular
Form) after cotransfection to HEK293 cells, the HEK293 cell mass (colony) resistant to homomycin (hygromycin)
Quantity；
Figure 1B is a block diagram, be about will be sheared with enzyme/helper plasmid for preparing of connection method and DNA donors it is (linear
Form) after cotransfection to HEK293 cells, the quantity of the HEK293 cell mass resistant to homomycin；
Fig. 1 C are the block diagram that context implementation 1 is illustrated according to the present invention, are about will be with commercial kit system
After standby helper plasmid and DNA donors cotransfection to HEK293 cells, the number of the HEK293 cell mass resistant to homomycin
Amount；
Fig. 2A is a block diagram, be about by sheared with enzyme/connection method prepare helper plasmid and DNA donor cotransfections
To Jurkat T cells, the quantity of the Jurkat T cell group resistant to homomycin；
Fig. 2 B are the block diagram that context implementation 2 is illustrated according to the present invention, are about will be with commercial kit system
After standby helper plasmid and DNA donors cotransfection to Jurkat T cells, the Jurkat T cell group resistant to homomycin
Quantity；And
Fig. 3 is the block diagram that context implementation 3 is illustrated according to the present invention, is about specific DNA donors and auxiliary matter
Grain indexable effect caused by primary human T cells.
Specific implementation mode
In order to keep the narration of the content of present invention more detailed with it is complete, below for embodiments of the present invention with it is specific
Embodiment proposes illustrative description；But this not implements or uses the unique forms of the specific embodiment of the invention.Embodiment party
The feature of multiple specific embodiments is covered in formula and to construction and the method and step for operating these specific embodiments and its
Sequentially.However, other specific embodiments can also be used to reach identical or impartial function and sequence of steps.
1. definition
Unless this specification is defined otherwise, meaning and those skilled in the art institute of science and technology vocabulary used herein
Understand identical as usual meaning.In addition, in the case of getting along well context conflict, the singular noun used in this specification covers
The complex number type of the noun；And the singular type of the noun is also covered by when used plural noun.Specifically, in this specification and power
In sharp claim, singulative " one " (a) and " one " (an) include plural reference value, but person indicated otherwise removes according to context
Outside.In addition, in this specification and claims, the meaning of form of presentation such as " at least one " and " one or more " is identical, two
Person represents and contains one, two, three or more.
Although to define the numerical value that the numberical range of wider range of the present invention and parameter are all rough, herein as far as possible
The correlation values in specific embodiment are accurately presented.However, any numerical value substantially inevitably contains because of individual tests
Standard deviation caused by method.Here, " about " typically refer to actual numerical value a certain number value or range positive and negative 10%,
5%, within 1% or 0.5%.Either, " about " word represents actual numerical value and falls within the acceptable standard error of average value,
Depending on depending on those skilled in the art the considerations of.Other than experimental example, or unless explicitly stated, when being appreciated that this place
All ranges, quantity, numerical value and percentage are (such as to describe material utilization amount, time length, temperature, operating condition, number
Amount ratio and other similar persons) by the modification of " about ".Therefore, unless otherwise opposite explanation, this specification and subsidiary power
Numerical parameter disclosed in sharp claim is all rough numerical value, and visual demand and change.It at least should be by these numerical parameters
It is interpreted as pointed number of significant digit and applies mechanically the obtained numerical value of general transfer method.
In holding within the present invention, " transposon " (transposon or transposable element) word refers to more than one
Nucleotide is integrated into a target position (for example, the base of cell after being cut by a donor polynucleotide (for example, a carrier)
Because of group or extra-chromosome (extrachromosome)), to change its position in genome.Transposon is to include a core
Two ends of the polynucleotides of nucleotide sequence, the wherein nucleotide sequence are respectively provided with cis acting (cis-acting) nucleotides sequence
Row, wherein at least a cis-acting nucleotide sequence are positioned at the ends 5' of the nucleotide sequence, and an at least cis acting nucleosides
Acid sequence is positioned at the ends 3' of the nucleotide sequence.The cis-acting nucleotide sequence respectively held positioned at transposon includes at least one anti-
To repetition (inverted repeat, IR)；Translocase (the preferably translocase of mammal piggyBac families) is in combination with extremely
The inverted repeat.In a better embodiment, transposon is the micro-piggyBac transposons from moth.
In holding within the present invention, " translocase " (transposase) word refers to a polypeptide, can be catalyzed more by a donor
Nucleotide (such as a micro-loop (minicircle) construct) is cut out the reaction of transposon, and the transposon is then integrated into one
In the genome or extra-chromosome DNA of target cell.Preferably, translocase can be bound to inverted repeats.
In holding within the present invention, " polypeptide " (polypeptide) word refers to that the amino acid with any length polymerize
Object.Therefore, the definition of polypeptide includes peptide (peptide), oligopeptides (oligopeptide), albumen (protein), antibody
(antibody) and enzyme (enzyme)." polypeptide " (polypeptide) word also include polypeptide translation after modify, citing come
It says, candy (glycosylation, a such as carbohydrate is added), acetylation (acetylation) and phosphorylation
(phosphorylation) etc..
" carrier " (vector) word refers to a nucleic acid molecules in this specification, the transmittable another nucleic acid linked with it
Molecule.Illustrative carrier include, but are not limited to, bacterium, plasmid, bacteriophage, clay (cosmid), episome (episome),
Virus and pluggable DNA fragmentation, such as can be thin by homologous recombination (homologous recombination) insertion host
The segment of the genome of born of the same parents.
In the present specification, " plasmid " (plasmid) word refers to cricoid distrand DNA, is subjected to an external DNA pieces
Section, and can be replicated in protokaryon or eukaryotic.
" micro-loop " (minicircle), " minicircle dna " (minicircle DNA) and " micro-loop nucleotide sequence "
(minicircle nucleic acid sequence) this specification be interchangeable vocabulary, all refer to one do not include it is any
The nucleotide sequence of plasmid/carrier framework needed for replicating, such as the antibiotics resistance gene of prokaryotes and prokaryotes
Replication origin.Can be to utilize the intramolecular between recombinase identification site in plasmid with bacterial plasmid in vivo preparing micro-loop
It recombinates to generate a minicircle dna carrier for not having bacterial plasmid backbone DNA.One embodiment of content according to the present invention is profit
Sheared with enzyme/connection method prepares micro-loop of the present invention.Another embodiment of content according to the present invention is to utilize commercial kit
(minicircle dna reagent preparation box, System Biosciences, CA, USA) prepares micro-loop of the present invention.
In holding within the present invention, " being transferred to " (introduce) word refers to by a polynucleotides (such as transposon of the present invention
The micro-loop nucleotide sequence or assistant carrier of system) it is transferred to a cell or organism.The nucleic acid of polynucleotides can be exposed
DNA or RNA, the DNA or RNA connected from different albumen, or it is inserted into the DNA or RNA of a carrier." being transferred to " (introduce) one
Word holds within the present invention covers widest range；For example, it is transferred to the transmission effect reached comprising following methods：Turn
Dye method (polynucleotides are transferred to an eukaryocyte by transfection using physics and/or chemical treatment), conversion method
(polynucleotides are transferred to a prokaryotic cell by transformation method using physics and/or chemical treatment), virus
Method/viral transduction method (viral method/viral transduction method, with virus or viral vectors by a multinuclear
Thuja acid is transferred to an eukaryon and/or prokaryotic cell), (conjugation method, are in direct contact bonding method by cell-ECM
Or intercellular cytoplasmic bridge, a polynucleotides are transferred to another cell by a cell) and fusion method (fusion method, melt
Two cells are closed, including homocellular fusion and heterocyst fusion).
It refers to one cell of processing that " transformation " (engineer) holds within the present invention, and the cell is caused to generate the change that can be detected
Change, the wherein processing include, but are not limited to, and be inserted into one with cell heterologous (heterologous)/homologous (homologous's)
Polynucleotides and/or polypeptide, and the polynucleotides and/or polypeptide that keep a cell primary generate mutation.
In holding within the present invention, " indexing " (transposition) word refers to a complicated genetic recombination process, including
One polynucleotides (transposon) are moved or copied into another site by a site, which often betides in genome, base
Because between group, between DNA construct (such as plasmid, rod granule (bacmid) and clay) or genome and DNA construct it
Between.Content Implementation mode according to the present invention, indexing are betided between genome and DNA construct, wherein polynucleotides (such as
The expression group of indexing subsystem of the invention) it is that host cell is transferred to by the micro-loop nucleotide sequence of indexable subsystem of the invention
The genome of (such as immunocyte).
It refers to the packet in a group host cell that " indexable effect " (transposition efficacy) holds within the present invention
Quantity containing the host cell for being transferred to polynucleotides.It in general, can be by one to the more of encoding reporter protein (such as β-gal)
Nucleotide, which is transfected, determines indexable effect to target cell.Accordingly, can by analysis be transferred to polynucleotides gene outcome (such as
Detection has effects that the active cell quantities of β-gal) it is indexable to determine.One embodiment of content according to the present invention, what is be transferred to is more
Nucleotide is a homomycin resistant gene, therefore can determine indexable work(by detecting the cell quantity resistant to homomycin
Effect.
In holding within the present invention, " immunocyte " (immune cell) word refers to the cell that can influence immune response.Exempt from
Epidemic disease cell is derived from the cell of haematological origin, including lymphocyte (such as B cell and T cell), constant killer cell and medullary substance
Property cell (such as monocyte (monocyte), macrophage (macrophage), acidophic cell (eosinophil), fertilizer
Maxicell (mast cell), basicyte (basophil) and granulocyte (granulocyte)).
Within the present invention hold in, " immune correlated disease " (immune-related disease) refer to a disease and/or
Symptom, wherein immune system relate to the pathogenic mechanism of the disease, or the stimulation or inhibition of appropriateness can treat and/or prevent this
Disease.The immune correlated disease of illustration formula include, but are not limited to, tumour, infectious diseases, allergy, autoimmune disease, transplanting
Object versus-host disease (graft-versus-host disease) or inflammatory diseases.
" individual " (subject) word refers to the animal for including the mankind, is subjected to the treatment of the method for the present invention.Unless special
Surely it points out, otherwise " individual " (subject) word means male and women simultaneously.
2. invention describes
The content of present invention be about to engineered cells, make cell carry specific gene (such as gene for the treatment of albumen)
System, method and/or kit.Cell after engineered can be used as a kind of healing potion, according to this to acceptable specific gene production
The disease and/or symptom of object treatment generate therapeutic efficiency.
It can be in an exogenous DNA fragmentation be in vitro integrated into a cell as described above, the present disclosure provides one kind
Genome method.The DNA sequence dna codified goes out the resistant albumen (antibiotic of a pair of of antibiotic
Resistance protein), a siRNA, a reporter protein (reporter protein), a cytohormone
(cytokine), a kinases (kinase), an antigen (antigen), a receptor (antigen- with antigentic specificity
Specific receptor), a cytokine receptor (cytokine receptor) or a suicide polypeptide (suicide
polypeptide).For example, foreign DNA sequences codified goes out a kind of to tumor associated antigen (tumor-
Associated antigen) receptor with specificity.Via the improved T cell of the method for the present invention, it can recognize and have specially
Kill the tumour cell that can express the tumor associated antigen to one property.In another embodiment, foreign DNA sequences codified goes out
A kind of albumen resistant to homomycin, to establish the cell strain resistant to homomycin.Either, exogenous DNA sequences
Row can not have any biological function, but by the way that itself segment to be inserted into an essential gene, to block the function of another gene.
For example, foreign DNA sequences codified goes out an antisense RNA (anti-sense RNA), with keep silent PD-1 or T cell it is special
The gene expression of receptor (T cell specific receptor, TCR).
First, an indexable subsystem is obtained to execute the method for the present invention.
The indexing subsystem includes a first vector, is had by SEQ ID No comprising one:1 nucleotide formed
First inverted repeat of sequence, one is exogenous positioned at this positioned at the exogenous DNA fragmentation in the first inverted repeat downstream and one
DNA sequence dna downstream, and with by SEQ ID No:Second inverted repeat of 2 nucleotide sequences formed.The first vector can
Further include a non-promoter in prokaryote, be operatively be connected to the foreign DNA sequences.For example, the non-original
Core biology promoter can be cytomegalovirus (cytomegalovirus, CMV) promoter, Rous sarcoma virus (rous
Sarcoma virus, RSV) promoter, simian virus (simian virus, SV40) promoter, mouse mammary tumour virus (mouse
Mammary tumor virus, MMTV) promoter, phosphoglycerokinase (phosphoglycerate kinase, PGK) start
Active (chicken beta-active) promoter of son, chicken second type, elongation factor 1- α (elongation factor 1-
Alpha, EF1- α) promoter, mankind H1 promoters (human H1promoter) or U6 promoters (U6promoter).One
In embodiment, which further includes an enhancer (enhancer), a silencer (silencer) or an insulator
(insulator)。
In a particular implementation, which is one not comprising bacterium prokaryotes sequence required when replicating
Minicircle dna.After excluding foreign DNA sequences, the sequence length of the minicircle dna is 500-1,500bp.For example, exist
After excluding foreign DNA sequences, the sequence length of the minicircle dna can be 700-1,200bp or 800-1,000bp.
In general, prokaryotes DNA is often considered as a kind of immunostimulating antigen, can cause host and generate immune response
And reduce expression effect of the exogenous DNA of first vector carrying.Therefore, compared to other comprising prokaryotes DNA sequence dna
For transposon/expression system, lacking the minicircle dna of prokaryotes sequence can make indexable subsystem of the invention efficiently will
Exogenous DNA is transferred to host cell (such as an Eukaryotic cell) and is expressed.In addition, compared to traditional transposon/
Expression system, present invention indexing subsystem have smaller volume/length since minicircle dna lacks prokaryotes DNA sequence dna.
Indexing subsystem of the invention also includes a Second support (for example, a helper plasmid), it includes an operatively
It is connected to the promoter of a nucleic acid, wherein the nucleic acid codified goes out a translocase.The translocase can recognize and be bound to the first load
The first inverted repeat and the second inverted repeat of body.For example, the translocase can be ThyPLGMH, mycPBase,
TPLGMH or HAhyPBase.In a particular implementation, which has SEQ ID No:4 amino acid sequence is
By SEQ ID No:3 encode and obtain.It can be according to method well-known to those skilled in the art or according to Yaa-Jyuhn James
Meir et.al.(A versatile,highly efficient,and potentially safer piggyBac
transposon system for mammalian genome manipulations,FASEB,2013:27,4429-4443)
The method prepares helper plasmid.
Promoter positioned at the Second support is selected from by cytomegalovirus promoter, rous sarcoma virus promoter, ape
Monkey disease virus promoter, mouse mammary tumor virus promoter, phosphoglycerokinase promoter, chicken second type promoter active, extend because
The group that sub- 1- α promoters, mankind H1 promoters and U6 promoters are formed.In a particular implementation, promoter is huge
Cell virus promoter.
It is that first vector and Second support are transferred in a cell when executing the method for the present invention.The illustrative side of being transferred to
Method include, but are not limited to, coprecipitation of calcium phosphate (calcium phosphate co-precipitation), electroporation
(electroporation), nuclear transfection (nucleofection), cell squeeze (cell squeezing, gently press cell membrane),
Ultrasonic perforation (sonoporation forms hole using high intensity ultrasonic in cell membrane), light transfect (optical
Transfection, using height focus laser in cell membrane manufacture a small hole), gene deliver (impalefection, will
DNA is connected to a nanofiber surface and injects in cell), (DNA is connected to an inert solid to particle gun by gene gun
After nano-particle, inject target cell core in), magnetic transfection (magnetofection is sent DNA to target cell using magnetic force
It is interior), virus transfection (viral transduction are sent DNA to target cell using virus as carrier) is or with branch
Shaped polymer (dendrimer), liposome (liposome) and cationic polymer (cationic polymer) are transfected.
In one embodiment, be using non-liposomal chemical method (HD is transfected) indexable subsystem is transferred to cell
In.In another embodiment, it is that indexable subsystem is transferred in cell using nuclear transfection.In a particular implementation of the invention
In, it is that first first vector by indexing system carries out after threadinessization handle (linearize, such as use restriction enzyme), then is transferred to
Cell.
The weight ratio of first vector and Second support when being transferred to cell is about 2:1 to 1:1.One embodiment of foundation,
When exogenous DNA is transferred to an epithelial cell, first vector (such as minicircle dna) and the weight ratio of Second support are about 2:1 arrives
1:1.According to another embodiment, (immortal T cell), micro-loop core when exogenous DNA is transferred to an immortalization T cell
The weight ratio of nucleotide sequence and helper plasmid is about 2:1 to 1:1.According to yet another embodiment, exogenous DNA is being transferred to one
When primary T cell, the weight ratio of micro-loop nucleotide sequence and helper plasmid is about 2:1 to 1:1.
For Second support after cell expresses translocase, which can be catalyzed is cut out exogenous DNA sequences by first vector
The reaction of row, and be integrated into the exogenous DNA come is cut out in the genome of cell.
Cell used in the method for the present invention can be an immunocyte.More specifically, which it is thin to can be T
Born of the same parents, B cell, dendritic cells (dendritic cell), macrophage or mast cell.
In another embodiment, which is a stem cell.For example, stem cell can be originated from marrow (bone
Marrow), adipose tissue (adipose tissue), peripheral blood (peripheral blood), Cord blood (umbilical
Cord blood) or dental pulp (dental pulp).In an ad hoc approach, which is a human cell.
To execute the above method, the content of present invention, which also provides one, can be integrated into foreign DNA sequences the genome of cell
Kit.The kit includes a container, and it includes aforementioned present invention indexing subsystem to be；And one accompanying operation say
It is bright, to indicate how using the indexable subsystem of the present invention.As described above, present invention indexing subsystem includes one first and one second
Carrier.
First vector includes one with by SEQ ID No:First inverted repeat of 1 nucleotide sequence formed, one
In the first inverted repeat downstream and with by SEQ ID No:Second inverted repeat of 2 nucleotide sequences formed, and
One between first inverted repeat and second inverted repeat, cloning site that foreign DNA sequences can be made to be transferred to.
The first vector of kit of the present invention can further include the promoter of a non-prokaryotes, be to be located at the first reversed weight
The upstream in multiple downstream and cloning site.After exogenous DNA is inserted into cloning site, the promoter of non-protokaryon animal can be grasped
It is connected to the foreign DNA sequences with making formula.For example, the promoter of non-prokaryotes can be that cytomegalovirus starts
Son, rous sarcoma virus promoter, simian virus promoter, mouse mammary tumor virus promoter, phosphoglycerokinase promoter, chicken
Second type promoter active, elongation factor 1- α promoters, mankind H1 promoters or U6 promoters.In one embodiment, it first carries
Body further includes an enhancer, a silencer or an insulator.
Kit of the present invention includes also a Second support, i.e. a helper plasmid is connected to comprising an operatively
The promoter of one nucleic acid, wherein the nucleic acid codified go out a translocase.For example, the translocase can be ThyPLGMH,
MycPBase, TPLGMH or HAhyPBase.In a particular implementation, which has SEQ ID No:4 amino acid
Sequence.
As described in the method for the present invention, the Second support of kit of the present invention includes a promoter, is selected from by giant cell
Viral promotors, rous sarcoma virus promoter, simian virus promoter, mouse mammary tumor virus promoter, phosphoglycerokinase
The group that promoter, chicken second type promoter active, elongation factor 1- α promoters, mankind H1 promoters and U6 promoters are formed
Group.In a particular implementation, which is cytomegalovirus promoter.
In holding within the present invention, " using operating instruction " (instruction) word contains book, records object, chart
Or any media (such as tape, CD, VCD or DVD), it can be used to inform or indicate user how using present invention indexing
Subsystem.It is securable on container using operating instruction, or is separately packed with the container comprising indexable subsystem of the invention.
Kit of the present invention can further include one to stablize indexable subsystem and/or carry out the buffer solution of cell transfecting.
For example, buffer solution can be phosphate-buffered normal saline solution (phosphate-buffered saline, PBS), packet
Normal saline solution (Tris-based saline), Tris-EDTA buffer solutions, 4- (2- ethoxys) -1- piperazine second sulphurs containing Tris
Sour (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer solution or bis- (the 2- hydroxyl second of N, N-
Base) -2- amidos ethanesulfonic acid (N, N-bis (2-hydroxyethyl) -2-amino ethanesulfonic acid, BES) buffering
Liquid.
As described above, being suffered from or the doubtful individual for suffering from immune correlated disease the present disclosure provides a kind of to treat
Method.This method includes that a cell is transformed using kit of the present invention, so that the cell is carried one immune suitable for treating this
The gene of relevant disease；And an a effective amount of modified cell is administered to the individual, to treat the immune correlated disease.
In one embodiment, gene is the Chimeric antigen receptor (chimeric antigen receptor, CAR) that can recognize CD19, can be led to
Transformation CAR T cells are crossed to treat acute lymphatic leukemia.
It suffers from or the doubtful individual for suffering from immune correlated disease is a mammal, including the mankind, mouse, rat, rabbit
Son, monkey and pig.In a particular implementation, individual is the mankind.When the individual for the treatment of is the mankind, the warp of the method for the present invention
The immunocyte for being transferred to/transfecting preferably is originated from the individual itself.Either, the method for the present invention is immune through what is be transferred to/transfect
Cell is derived from a donor.
Content certain embodiments according to the present invention, the method for the present invention can be used to treat an immunosuppressant disease, such as swollen
Tumor or infectious diseases.In these embodiments, indexing system of the present invention may include an Immune-enhancing effect gene, and using promotion should
The immune response of individual.
Content certain embodiments according to the present invention, the method for the present invention can be used to treat one because of immune response overactivity institute
Caused by disease (such as autoimmune disease) or disease (such as the anti-place of allergy, graft caused by immunoreaction abnormity
Main disease or inflammatory diseases).In these embodiments, indexing system of the present invention may include an immune suppressor genes, with suppression
Make the immune response of the individual.
Multiple experimental examples are presented below to illustrate the certain embodiments of the present invention, this is operated with sharp those skilled in the art
Invention, and these experimental examples should not be considered as limitation of the scope of the invention.It is herein proposed according to technical staff having read
After bright, it can completely utilize in the case of being not required to excessively understand and put into practice the present invention.All open source literatures cited herein,
Its full text is all considered as the part of this specification.
Embodiment
Material and method
Cell culture
This research is to immortalize T lymph corpuscles (Jurkat using Human embryonic kidney's cell strain 293 (HEK 293), mankind JK
T) and primary human T cells carry out correlation analysis.By HEK293 cell culture in including 10% fetal calf serum, 2mM L- glutamy
Amine (L-glutamine), 1 times of non-essential amino acid (nonessential amino acid), 1 times of penicillin/streptomycin
(penicillin/streptomycin) and in the MEM cell culture fluids of 1mM Sodium Pyruvates (sodium pyruvate).It will
Jurkat T cells and primary human T cells are incubated at comprising 2mM L-Glutamines, 10% fetal calf serum, 1mM Sodium Pyruvates
And in the RPMI1640 cell culture fluids of 0.1mM non-essential amino acids.All cells are all incubated at comprising 5%CO237 DEG C
In environment.
Preparation condition culture solution (conditioned medium)
By Jurkat cell with every milliliter of 2x 106The density of a cell is cultivated 24 hours in fresh cell medium, it
Simultaneously filtration cell culture solution is collected afterwards, as conditioned medium.
Prepare plasmid and/or expression construct
pBS-cassette
It is cut out by pcDNA3.1_hygro_LacZ carriers (Invitrogen) even mould comprising being started by SV40 promoters
The DNA fragmentation of plain resistant gene.After being sheared with XmnI and SapI enzymes, DNA fragmentation is cloned into pBlueScript SKII's
Construction pBS-hygro is used in the positions SmaI.For be further inserted into the gene resistant to kanamycins (kanamycin) and
ApoI_AflIII segments in pZErO-2.1 (Invitrogen) are cloned into pBS-hygro's by replication origin ColE1
The positions EcoRV, with construction pBS-cassette.
Mini-piggyBac_long (long formula mini-piggyBac)
After restriction enzyme SmaI and EcoRV shearing pBS-cassette, segment will be sheared and be inserted into from pBSII-ITR1
In pXLBacIIPUbnlsEGFP.Construct mini-piggyBac_long obtained 5 ' and 3 ' end be respectively provided with 308bp and
The end of 238bp repeats (terminal repeat, TR).
Construct mini-piggyBac_long is sheared using XmnI, to prepare linear mini-piggyBac_long.
Mini-piggyBac_short (short formula mini-piggyBac)
Mini-piggyBac_long constructs are sheared with PciI and AclI, after being handled with klenow enzymes (NEBiolabs),
Self connection reaction is carried out using T4DNA ligases (NE Biolabs), is prepared according to this not comprising An Benzyl penicillin
(ampicillin) mini-piggyBac_short of resistant gene and f1 replication origins.
Mini-piggyBac_short is sheared to prepare linear mini-piggyBac_short using BglI.
Micro-piggyBac_long (long formula micro-piggyBac)
The terminal repeats short formula piggyBac domain is made in the PCR mixtures that are formed using following four pairs of introductions
(terminal repeat domain, TRD) (i.e. 746~808 3 ' LTR and 1426~1,460 5 ' in pXL-BacII
LTR)：pB-11-KpnI(SEQ ID No:5), pB-5- forward directions (SEQ ID No:6), reversed (the SEQ ID No of pB-6-:7) and
pB-12-SacI(SEQ ID No:8).The amplification of 67bp 5 ', 40bp 3 ' TRD and SwaI and XhoI restriction enzymes position will be included
Sub (amplicon), is cloned into pBS-SKII with Kpn I and Sac I, to prepare pPBendAATT.It will be by above-mentioned with Xho I
The expressing gene group that pBS-cassette is obtained is inserted between the short formula piggyback TRD of pPBendAATT, to prepare
micro-piggyBac_long。
Micro-piggyBac_long is sheared using XmnI, to prepare linear micro-piggyBac_long.
Micro-piggyBac_short (short formula micro-piggyBac)
Micro-piggyBac_long is sheared with Acc65I and AflIII, to remove An Benzyl ampicillin resistance genes and f1
Replication origin.Make self connection of remaining DNA fragmentation, to prepare not amine-containing Benzyl ampicillin resistance genes and f1 replication origins
micro-piggyBac_short.The construct is also referred to as micro-loop genome (minicircle cassette).
Micro-piggyBac_short is sheared using XmnI, to prepare linear micro-piggyBac_short.
Minicircle-microPB-cassette
Either, micro-piggyBac_short can be prepared using MC-Easy ring-types minicircle dna reagent preparation box
(i.e. micro-loop genome).Such method is after shearing micro-piggyBac_long with KpnI, SacI and XmnI, will to include
The KpnI-SacI segments (3993bp) an of left side (microL) of micro-piggyBac and right (microR) inverted repeat are inserted into
The EcoRV restriction enzymes position of pMC.BESPX-MCS1, to prepare pMC-microPB-cassette.According to using operating instruction,
Minicircle- is prepared by pMC-microPB-cassette with MC-Easy circle ring-type minicircle dna reagent preparation boxes
microPB-cassette.Minicircle-microPB-cassette obtained do not include ampicillin resistance gene and
F1 replication origins.
Helper plasmid
According to Yaa-Jyuhn James Meir et.al. (A versatile, highly efficient, and
potentially safer piggyBac transposon system for mammalian genome
manipulations,FASEB,2013:27,4429-4443) the method come construction helper plasmid pCMV-ThyPLGMH,
PCMV-mycPBase, pCMV-TPLGMH and pCMV-HAhyPBase.The full text of the displosure document is considered as the one of the content of present invention
Part.
Indexing experiment
HEK293 cells
By eighty per cant full cell seeding in 24 porose discs, cell density is per hole 1x 105A cell.Cell is carried out to turn
When dye, the DNA mixtures of 300 nanograms are transfected into cell with Fugene 6 (Roche, Florence, SC).Each DNA mixing
Object includes helper plasmid (i.e. pCMV-ThyPLGMH, pCMV-mycPBase, pCMV-TPLGMH or pCMV- of 100 nanograms
HAhyPBase), different amounts of DNA donors (i.e. mini-piggyBac_long, mini-piggyBac_short, micro-
PiggyBac_long, micro-piggyBac_short, pMC-microPB-cassette or minicircle-microPB-
cassette；Minimum is 100 nanograms for the scale of construction), and make final DNA content up to the pcDNA3.1 of 300 nanograms.After transfection,
/ 15th cell through transfection is taken to be screened 14 days to 100 millimeters of disks, then with homomycin.Using including 4% metaformaldehyde
(paraformaldehyde) PBS is fixed after ten minutes, is dyed 1 hour with 0.2% methylenum careuleum (methylene blue), with
Count cell mass.After homomycin screens 14 days, only calculated diameter is 0.5 millimeter of cell mass.
Jurkat T cells
By every milliliter of 1x 106After a cell culture 24 hours, nuclear transfection is carried out.Each nuclear transfection reaction is all by 6 micrograms
DNA mixtures are transfected to 1x 106In a cell.Each DNA mixtures include helper plasmid (the i.e. pCMV- of 2.5 micrograms
ThyPLGMH, pCMV-mycPBase, pCMV-TPLGMH or pCMV-HAhyPBase), different amounts of DNA donors (i.e. mini-
piggyBac_long、mini-piggyBac_short、micro-piggyBac_long、micro-piggyBac_short、
PMC-microPB-cassette or minicircle-microPB-cassette；Minimum is 2.0 micrograms for the scale of construction), and
Make final DNA content up to the pcDNA3.1 of 6 micrograms.After nuclear transfection 24 hours, 50 microlitres of above-mentioned condition trainings are suspended in by 30
The living cells of nutrient solution is screened with 1.2 milligrams of homomycin in 96 porose discs.Every 2 to 3 days, it is slowly added to isometric condition
Culture solution avoids influencing cell mass.Each reaction is screened with 10,200 living cells of homomycin pair.It is screened through homomycin
After 6 or 7 days, the quantity of the cell mass of each reaction is calculated using light microscope, to obtain its indexing activity.
Primary T cell
Nuclear transfection the previous day is being carried out, at the beginning of the full cell culture fluids of RPMI (RPMI complete medium) the defrosting mankind
For cell (human primary cell, CD8+CD45RA+).Culture to the next day after, to cell carry out nuclear transfection.Each nuclear transfection
Reaction is all to transfect the DNA mixtures of 2.2 micrograms to the ALL-IN-ONE nuclear transfection buffer solutions for being suspended in 20 microlitres
The 1X10 of (GF1001, GenomeFrontier, Biosciences)5In a cell.Each DNA mixtures include the auxiliary of 0.8 microgram
Help plasmid (i.e. pCMV-HAhyPBase or pCMV-ThyPLGMH), different amounts of donor plasmid (i.e. mini-piggyBac_
Long, mini-piggyBac_short, micro-piggyBac_long or micro-piggyBac_short；Maximum donor
Amount is 1.4 micrograms), and make final DNA content up to the pcDNA3.1 of 2.2 micrograms.After nuclear transfection 24 hours, with 500 microlitres
Include the 1640 full cell culture of RPMI of stimulant (PHA and the IL-2 of every milliliter of 50 micrograms) and every milliliter of 1.0 milligrams of homomycin
Cell of the liquid culture through transfection.After homomycin screens 22 days, calculating survivaling cell number is to obtain indexable activity.
Embodiment 1 is in the indexing activity of HEK293 cells
The present embodiment will analyze each DNA donors and helper plasmid in the indexing activity of HEK293 cells.As a result it illustrates respectively
In Figure 1A -1C.
As shown in Figure 1A, though the DNA donors of cotransfection why, compared to pcDNA3.1, pCMV-TPLGMH or
The cell that pCMV-mycPBase is transfected, the cell transfected through pCMV-ThyPLGMH, which can be formed, most to be had homomycin
The cell mass of resistance.The result points out that ThyPLGMH is the highest translocase of indexing activity in test translocase.It is supplied as DNA
Body, DNA donors (i.e. mini-piggyBac_long, mini-piggyBac_short, micro-piggyBac_ of four kinds of tests
Long and micro-piggyBac_short) have similar indexing active (Figure 1A) in HEK293 cells.Make us ground of being surprised,
When DNA donors are transfected to HEK293 cells with its string-like form, micro-piggyBac (i.e. micro-piggyBac_
Short or micro-piggyBac_long) indexable activity can be significantly higher than mini-piggyBac (i.e. mini-
PiggyBac_short or mini-piggyBac_long) indexing it is active (Figure 1B), wherein micro-piggyBac_short
With highest indexing activity.
Further detection is with the indexing activity of the micro-loop obtained by MC-Easy ring-type minicircle dna reagent preparation boxes.Fig. 1 C's
As a result it points out, when with pCMV-ThyPLGMH cotransfections, the activity of minicircle-microPB-cassette is pMC-
Active 2.7 times of microPB-cassette.
On the whole, these results point out that indexable activity can be as DNA donors overall length or the length of its TRD increase and subtract
It is few；Compared to the translocase of other tests, ThyPLGMH has highest indexing activity.Therefore, compared to other DNA donors
And helper plasmid, pCMV-ThyPLGMH and micro-piggyBac micro-loops (i.e. micro-piggyBac_short or
Being combined in HEK293 cells minicircle-microPB-cassette) can generate highest indexable effect.
Embodiment 2 is in the indexing activity of Jurkat T cells
Then the indexing activity of pCMV-ThyPLGMH and micro-piggyBac micro-loops is detected in Jurkat T cells.
As a result it is set forth in Fig. 2A -2B respectively.
As shown in Figure 2 A, compared to the cell of other DNA donors and helper plasmid cotransfection, by pCMV-ThyPLGMH
And the cell of micro-piggyBac_short cotransfections can generate the homomycin resisting cell group of significantly higher amount.
Present similar with Fig. 1 C as a result, combine compared to other DNA donors/helper plasmids, pCMV-ThyPLGMH and
The combination of minicircle-microPB-cassette can generate highest indexable effect (Fig. 2 B).
Embodiment 3 is in the indexing activity of primary human T cells
Other than above-mentioned cell strain (i.e. HEK293 cells and Jurkat T cells), also divided using primary human T cells
Analyse the indexing activity of specific DNA donors and helper plasmid.
As shown in figure 3, in through pCMV-ThyPLGMH and short formula or long formula micro-piggyBac (i.e. micro-
PiggyBac_short or micro-piggyBac_long) best indexing activity can be observed in the cell of transfection.
Summarize above-mentioned, the present disclosure provides a kind of transposon system and methods, can be used to an allogenic gene is whole
It is bonded in the genome of a cell (an especially immunocyte).Compared to the combination of other DNA and helper plasmid, pCMV-
ThyPLGMH and micro-piggyBac micro-loops (micro-piggyBac_short whether prepared by enzyme shearing/connection method,
Or the minicircle-microPB-cassette made from MC-Easy ring-type minicircle dna reagent preparation boxes) or micro-
The combination of piggyBac_long can generate highest indexable effect.Therefore, the content of present invention additionally provides one kind to treat not
The method of same disease (such as immune correlated disease), be by by a therapeutic gene be effectively transferred to it is in need individual in vivo,
To achieve the purpose that treatment.
Although specific embodiments of the present invention are disclosed in embodiment above, those skilled in the art, when can to its into
The various changes of row and modification.Description above, embodiment and experimental data are to the content of present invention as illustrative embodiments
In structure and occupation mode done complete description.Although the embodiment that various kinds in the content of present invention has been described above has one
Determine the characteristic of degree, or with reference to one or more other embodiments, those skilled in the art remain to do not departing from the present invention
Hold under spirit and scope situation, numerous modifications are carried out to disclosed embodiment.
Sequence table
<110>Wu Qiongyuan
<120>Indexable subsystem, kit and application thereof
<130> 18FAP0132CPT
<150> US62/267,270
<151> 2015-12-14
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 67
<212> DNA
<213>Artificial sequence (artificial sequence)
<220>
<221> primer_bind
<223>Composition sequence -5-IR
<400> 1
ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg 60
acgcatg 67
<210> 2
<211> 40
<212> DNA
<213>Artificial sequence (artificial sequence)
<220>
<221> primer_bind
<223>Composition sequence -3-IR
<400> 2
gcatgcgtca attttacgca gactatcttt ctagggttaa 40
<210> 3
<211> 2697
<212> DNA
<213>Artificial sequence (artificial sequence)
<220>
<221> gene
<223>Composition sequence-ThyPLGMH
<400> 3
atgggtcgca agaaacgtcg ccaacgtcgc cgtccgccta tcatgactcg agccatgggc 60
agcagcctgg acgacgagca catcctgagc gccctgctgc agagcgacga cgagctggtc 120
ggcgaggaca gcgacagcga ggtgagcgac cacgtgagcg aggacgacgt gcagtccgac 180
accgaggagg ccttcatcga cgaggtgcac gaggtgcagc ctaccagcag cggctccgag 240
atcctggacg agcagaacgt gatcgagcag cccggcagct ccctggccag caacaggatc 300
ctgaccctgc cccagaggac catcaggggc aagaacaagc actgctggtc cacctccaag 360
cccaccaggc ggagcagggt gtccgccctg aacatcgtga gaagccagag gggccccacc 420
aggatgtgca ggaacatcta cgaccccctg ctgtgcttca agctgttctt caccgacgag 480
atcatcagcg agatcgtgaa gtggaccaac gccgagatca gcctgaagag gcgggagagc 540
atgacctccg ccaccttcag ggacaccaac gaggacgaga tctacgcctt cttcggcatc 600
ctggtgatga ccgccgtgag gaaggacaac cacatgagca ccgacgacct gttcgacaga 660
tccctgagca tggtgtacgt gagcgtgatg agcagggaca gattcgactt cctgatcaga 720
tgcctgagga tggacgacaa gagcatcagg cccaccctgc gggagaacga cgtgttcacc 780
cccgtgagaa agatctggga cctgttcatc caccagtgca tccagaacta cacccctggc 840
gcccacctga ccatcgacga gcagctgctg ggcttcaggg gcaggtgccc cttcagggtc 900
tatatcccca acaagcccag caagtacggc atcaagatcc tgatgatgtg cgacagcggc 960
accaagtaca tgatcaacgg catgccctac ctgggcaggg gcacccagac caacggcgtg 1020
cccctgggcg agtactacgt gaaggagctg tccaagcccg tccacggcag ctgcagaaac 1080
atcacctgcg acaactggtt caccagcatc cccctggcca agaacctgct gcaggagccc 1140
tacaagctga ccatcgtggg caccgtgaga agcaacaaga gagagatccc cgaggtcctg 1200
aagaacagca ggtccaggcc cgtgggcacc agcatgttct gcttcgacgg ccccctgacc 1260
ctggtgtcct acaagcccaa gcccgccaag atggtgtacc tgctgtccag ctgcgacgag 1320
gacgccagca tcaacgagag caccggcaag ccccagatgg tgatgtacta caaccagacc 1380
aagggcggcg tggacaccct ggaccagatg tgcagcgtga tgacctgcag cagaaagacc 1440
aacaggtggc ccatggccct gctgtacggc atgatcaaca tcgcctgcat caacagcttc 1500
atcatctaca gccacaacgt gagcagcaag ggcgagaagg tgcagagccg gaaaaagttc 1560
atgcggaacc tgtacatggg cctgacctcc agcttcatga ggaagaggct ggaggccccc 1620
accctgaaga gatacctgag ggacaacatc agcaacatcc tgcccaaaga ggtgcccggc 1680
accagcgacg acagcaccga ggagcccgtg atgaagaaga ggacctactg cacctactgt 1740
cccagcaaga tcagaagaaa ggccagcgcc agctgcaaga agtgtaagaa ggtcatctgc 1800
cgggagcaca acatcgacat gtgccagagc tgtttcaagc ttaagttggg cggcggcgcc 1860
cccgccgtgg gcggcggccc caaggccgcg gataaaccgg tcgccaccat ggtgagcaag 1920
ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 1980
ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc 2040
ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 2100
ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc 2160
ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac 2220
ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 2280
gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 2340
aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg 2400
aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag 2460
cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc 2520
cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 2580
gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagcttgg gcccgaacaa 2640
aaactcatct cagaagagga tctgaatagc gccgtcgacc atcatcatca tcatcat 2697
<210> 4
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35 40 45
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Phe Ile Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu
65 70 75 80
Ile Leu Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala
85 90 95
Ser Asn Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn
100 105 110
Lys His Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser
115 120 125
Ala Leu Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg
130 135 140
Asn Ile Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu
145 150 155 160
Ile Ile Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys
165 170 175
Arg Arg Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp
180 185 190
Glu Ile Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys
195 200 205
Asp Asn His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met
210 215 220
Val Tyr Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg
225 230 235 240
Cys Leu Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn
245 250 255
Asp Val Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln
260 265 270
Cys Ile Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln
275 280 285
Leu Leu Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn
290 295 300
Lys Pro Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly
305 310 315 320
Thr Lys Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln
325 330 335
Thr Asn Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys
340 345 350
Pro Val His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr
355 360 365
Ser Ile Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr
370 375 380
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385 390 395 400
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405 410 415
Gly Pro Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val
420 425 430
Tyr Leu Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr
435 440 445
Gly Lys Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val
450 455 460
Asp Thr Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr
465 470 475 480
Asn Arg Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys
485 490 495
Ile Asn Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu
500 505 510
Lys Val Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu
515 520 525
Thr Ser Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg
530 535 540
Tyr Leu Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly
545 550 555 560
Thr Ser Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr
565 570 575
Cys Thr Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys
580 585 590
Lys Lys Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys
595 600 605
Gln Ser Cys Phe Lys Leu Lys Leu Gly Gly Gly Ala Pro Ala Val Gly
610 615 620
Gly Gly Pro Lys Ala Ala Asp Lys Pro Val Ala Thr Met Val Ser Lys
625 630 635 640
Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp
645 650 655
Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly
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Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly
675 680 685
Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly
690 695 700
Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe
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Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe
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Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu
740 745 750
Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys
755 760 765
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785 790 795 800
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<210> 5
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<221> primer_bind
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<400> 5
atcgggtacc ttaaccctag aaagataatc atattg 36
<210> 6
<211> 75
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<213>Artificial sequence (artificial sequence)
<220>
<221> primer_bind
<223>Composition sequence-pB-5-forward
<400> 6
ggtaccccct agaaagataa tcatattgtg acgtacgtta aagataatca tgcgtaaaat 60
tgacgcatgc tcgag 75
<210> 7
<211> 76
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<213>Artificial sequence (artificial sequence)
<220>
<221> primer_bind
<223>Composition sequence-pB-6-reverse
<400> 7
gagctcccct agaaagatag tctgcgtaaa attgacgcat gccaccgcgg tggatttaaa 60
tctcgagcat gcgtca 76
<210> 8
<211> 34
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Claims (28)
1. a kind of method in the genome that a foreign DNA sequences are in vitro integrated into a cell, including：
(i) an indexable subsystem is provided, it includes：
First vector, including：
First inverted repeat has by SEQ ID No:1 nucleotide sequence formed,
The foreign DNA sequences are located at the downstream of first inverted repeat, and
Second inverted repeat is located at the downstream of the foreign DNA sequences, and with by SEQ ID No:2 cores formed
Nucleotide sequence；And
Second support, including operatively be connected to the promoter of a nucleic acid, wherein the nucleic acid, which can be used to encode, has SEQ
ID No:The translocase of 4 amino acid sequence；And
(ii) first vector of the indexable subsystem and the Second support are transferred in the cell, so that described exogenous
DNA sequence dna is integrated into the genome of the cell,
The wherein described translocase can be catalyzed described exogenous by being cut out in the first vector after the cell is expressed
The reaction of DNA sequence dna, and the clipped Exogenous Nucleic Acid is made to be integrated into the genome of the cell.
2. the method as described in claim 1, wherein to go out a pair of of antibiotic resistant for the foreign DNA sequences codified
Albumen, siRNA, reporter protein, cytohormone, kinases, antigen, the receptor with antigentic specificity, cytokine receptor or from
Kill polypeptide.
3. method as claimed in claim 2, wherein the first vector further includes non-promoter in prokaryote, operatively
It is connected to the foreign DNA sequences.
4. method as claimed in claim 3, wherein the non-promoter in prokaryote is selected from by cytomegalovirus promoter, labor
This Sarcoma Virus promoter, simian virus promoter, mouse mammary tumor virus promoter, phosphoglycerokinase promoter, chicken second
The group that type promoter active, elongation factor 1- α promoters, mankind H1 promoters and U6 promoters are formed.
5. method as claimed in claim 3, wherein the first vector further includes enhancer, silencer or insulator.
6. method as described in claim 1, wherein the promoter positioned at the Second support is selected from by cytomegalovirus
Promoter, rous sarcoma virus promoter, simian virus promoter, mouse mammary tumor virus promoter, phosphoglycerokinase start
The group that son, chicken second type promoter active, elongation factor 1- α promoters, mankind H1 promoters and U6 promoters are formed.
7. the either method as described in claim 1 to 6 does not include bacterium and replicates wherein the first vector is a minicircle dna
The prokaryotes sequence of Shi Suoxu, and after excluding the foreign DNA sequences, the length of the minicircle dna is 500-1,
500bp。
8. the method for claim 7, wherein after excluding the foreign DNA sequences, the length of the minicircle dna is
700-1,200bp。
9. method as claimed in claim 8, wherein after excluding the foreign DNA sequences, the length of the minicircle dna is
800-1,000bp。
10. the method as described in claim 1, wherein the cell is immunocyte, selected from by T cell, B cell, dendron
The group that cell, macrophage or mast cell are formed.
11. the method as described in claim 1, wherein the cell is stem cell, selected from by marrow, adipose tissue, periphery
The group that blood, Cord blood and dental pulp are formed.
12. the method as described in claim 1, further include it is described be transferred to step before threadinessization handle the first vector.
13. the method as described in claim 1, wherein the first vector and the Second support are to utilize following either method
It is transferred in the cell, the method is coprecipitation of calcium phosphate, electroporation, nuclear transfection, cell squeezes, ultrasonic is perforated, light turns
Dye, gene delivery, particle gun, magnetic transfection, virus transfection are turned with dendritic, liposome and cationic polymer
Dye.
14. the either method as described in claim 10 to 13, wherein the first vector is minicircle dna, it is multiple not comprising bacterium
Prokaryotes sequence needed for system, and after excluding the foreign DNA sequences, the length of the minicircle dna is 500-1,
500bp。
15. method as claimed in claim 14, wherein after excluding the foreign DNA sequences, the length of the minicircle dna
For 700-1,200bp.
16. method as claimed in claim 15, wherein after excluding the foreign DNA sequences, the length of the minicircle dna
For 800-1,000bp.
17. the method as described in claim 10 or 11, wherein the cell is human cell.
18. it is a kind of a foreign DNA sequences to be integrated into the kit of the genome of a cell, including：
Container；
Indexable subsystem, including：
First vector, including：
First inverted repeat has by SEQ ID No:1 nucleotide sequence formed,
Second inverted repeat has by SEQ ID No:2 nucleotide sequences formed, and positioned at first inverted repeat
Downstream, and
Cloning site can be used to be transferred to the external source between first inverted repeat and second inverted repeat
Property DNA sequence dna, and
Second support, including operatively be connected to the promoter of a nucleic acid, have wherein the nucleic acid can be used to encode one
SEQ ID No:The translocase of 4 amino acid sequence；And
It using operating instruction, is appended hereto in the container, and to indicate how using the indexable subsystem；
The wherein described translocase can be catalyzed described exogenous by being cut out in the first vector after the cell is expressed
The reaction of DNA sequence dna, and the clipped Exogenous Nucleic Acid is made to be integrated into the genome of the cell.
19. kit as claimed in claim 18, wherein the promoter positioned at the Second support is selected from by big and small
Cellular virus promoter, rous sarcoma virus promoter, simian virus promoter, mouse mammary tumor virus promoter, phosphoglycerol swash
What enzyme promoters, chicken second type promoter active, elongation factor 1- α promoters, mankind H1 promoters and U6 promoters were formed
Group.
20. kit as claimed in claim 18 is located at wherein the first vector further includes non-promoter in prokaryote
The downstream of first inverted repeat and the upstream of the cloning site, wherein the non-promoter in prokaryote can be selected from
By cytomegalovirus promoter, rous sarcoma virus promoter, simian virus promoter, mouse mammary tumor virus promoter, phosphoric acid
Glycerokinase promoter, chicken second type promoter active, elongation factor 1- α promoters, mankind H1 promoters and U6 promoters institute
The group of composition.
21. any kit as described in claim 18 to 20 does not include thin wherein the first vector is a minicircle dna
Bacterium replicates required prokaryotes sequence.
22. it is a kind of to treat one suffer from or it is doubtful suffer from an immune correlated disease individual method, including：(1) by making
An immunocyte is transformed with kit as claimed in claim 18, the immunocyte is made to carry an allogenic gene；With
And (2) administer a modified immunocyte of a effective amount of step (1) to the individual, to improve or reduce the immune correlation
The symptom of disease.
23. method as claimed in claim 22, wherein the individual is the mankind.
24. method as claimed in claim 22, wherein the immunocyte is derived from the individual or donor.
25. method as claimed in claim 22, wherein the allogenic gene can increase the immune response of the individual, and institute
It is tumour or infectious diseases to state immune correlated disease.
26. method as claimed in claim 22, wherein the allogenic gene can inhibit the immune response of the individual, and institute
It is allergy, autoimmune disease, graft versus host disease or inflammatory diseases to state immune correlated disease.
27. method as claimed in claim 22, wherein the immunocyte is T cell, B cell, dendritic cells, macrophage
Or mast cell.
28. method as claimed in claim 22, wherein to go out a pair of of antibiotic resistant for the foreign DNA sequences codified
Albumen, reporter protein, cytohormone, kinases, antigen, the receptor with antigentic specificity, cytokine receptor, suicide polypeptide
Or control component.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201562267270P | 2015-12-14 | 2015-12-14 | |
| US62/267,270 | 2015-12-14 | ||
| PCT/CN2016/109510 WO2017101749A1 (en) | 2015-12-14 | 2016-12-12 | Transposon system, kit comprising the same, and uses thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108474005A true CN108474005A (en) | 2018-08-31 |
| CN108474005B CN108474005B (en) | 2022-10-21 |
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| CN201680071463.4A Active CN108474005B (en) | 2015-12-14 | 2016-12-12 | Translocation subsystem, kit and use thereof |
Country Status (11)
| Country | Link |
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| US (1) | US11345926B2 (en) |
| JP (1) | JP6956416B2 (en) |
| KR (1) | KR102249982B1 (en) |
| CN (1) | CN108474005B (en) |
| AU (1) | AU2016373365B2 (en) |
| CA (1) | CA3004665C (en) |
| MX (1) | MX2018007203A (en) |
| MY (1) | MY184126A (en) |
| NZ (1) | NZ742068A (en) |
| TW (1) | TWI609960B (en) |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023060589A1 (en) * | 2021-10-15 | 2023-04-20 | 深圳市深研生物科技有限公司 | Multi-transposon system |
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| US11377651B2 (en) | 2016-10-19 | 2022-07-05 | Flodesign Sonics, Inc. | Cell therapy processes utilizing acoustophoresis |
| US20190119636A1 (en) | 2017-10-23 | 2019-04-25 | Poseida Therapeutics, Inc. | Modified stem cell memory t cells, methods of making and methods of using same |
| US11278570B2 (en) | 2016-12-16 | 2022-03-22 | B-Mogen Biotechnologies, Inc. | Enhanced hAT family transposon-mediated gene transfer and associated compositions, systems, and methods |
| EA201991431A1 (en) * | 2016-12-16 | 2020-01-17 | Б-Моген Биотехнолоджис, Инк. | REINFORCED MEDIUM-TRANSMISSED HAT FAMILY TRANSFER GENES AND ASSOCIATED COMPOSITIONS, SYSTEMS AND METHODS |
| US11760983B2 (en) | 2018-06-21 | 2023-09-19 | B-Mogen Biotechnologies, Inc. | Enhanced hAT family transposon-mediated gene transfer and associated compositions, systems, and methods |
| WO2023282730A1 (en) * | 2021-07-09 | 2023-01-12 | 주식회사 네오젠티씨 | Transposon system and uses thereof |
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| CA3004665A1 (en) | 2017-06-22 |
| KR102249982B1 (en) | 2021-05-11 |
| MX2018007203A (en) | 2018-11-12 |
| NZ742068A (en) | 2023-07-28 |
| JP2019503653A (en) | 2019-02-14 |
| US11345926B2 (en) | 2022-05-31 |
| TWI609960B (en) | 2018-01-01 |
| US20180320196A1 (en) | 2018-11-08 |
| CA3004665C (en) | 2021-08-24 |
| KR20180092989A (en) | 2018-08-20 |
| TW201720926A (en) | 2017-06-16 |
| JP6956416B2 (en) | 2021-11-02 |
| WO2017101749A1 (en) | 2017-06-22 |
| CN108474005B (en) | 2022-10-21 |
| MY184126A (en) | 2021-03-19 |
| AU2016373365B2 (en) | 2021-07-08 |
| AU2016373365A1 (en) | 2018-05-17 |
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